Recently, genome-wide screen and positional cloning for diabetes-susceptibility genes implicate that genetic variation in the CAPN10 gene, which encodes ubiquitously expressed cysteine protease calpain-10, is associated with increased risk of type 2 diabetes in a population of Mexican Americans who are susceptible to the disease, and also in an isolated population from Finland. Follow-up studies show, however, that levels of association between this putative diabetes-susceptibility gene and type 2 diabetes vary greatly in different populations. Whereas some studies have confirmed an association in African American, British, Polish, and South Indian populations, other investigations showed no association in other ethnic populations, including a large Finnish cohort, Japanese, and Scandinavian Caucasians. Clearly, the original hypothesis can be neither conclusively validated nor overturned without detailed biochemical studied. It is widely believed that identification of downstream substrates of calpain-10 holds the key to understand its action mechanisms and its relationship to type 2 diabetes. This proposal is directed at scanning the human proteome to identify the downstream targets of calpain-10 using a novel proteomic technology platform called mRNA display. First, human calpain-10 will be over-expressed using a baculovirus expression system and characterized. Then, human proteome domain libraries displayed on their own mRNAs are generated and immobilized on the solid surface via the biotin residue specifically introduced near the N-terminus of each protein. Upon incubation with purified calpain-10, protein sequences that are specifically cleaved by calpain-10 are released and enriched, with the intact mRNA still covalently attached to the C terminus of each cleaved protein fragment. The selected protein sequences are then regenerated for iterative round of selection, by PCR amplification followed by in vitro transcription/ translation, until the pool is dominated by sequences whose protein portions can be cleaved by calpain-10. The identity of each protein can be readily determined from its mRNA, by sequencing or cDNA microarray. The cleavage of selected proteins by calpain-10 will be studied using in vitro transcribed/ translated free proteins (or fragments) in the presence or absence of calpain inhibitors. Their cleavage sites will be mapped and kinetics will be studied. The project is expected to allow a systematic identification of potential physiological substrates for calpain-10 on a proteome-wide scale. The results will have significant implications in addressing the controversy of calpain-10 observed in different ethnic populations. The simplicity and high throughput of the technology will make the approach broadly used to scan the human proteome for downstream targets of other family members of calpains, which have been linked to other human pathological conditions, including Alzheimer's disease, neurological disorders, and gastric cancer.